Enzymatic method for the enantioselective reduction of keto compounds

ABSTRACT

The inventon relates to an enzymatic method for the enantioselective reduction of organic keto compounds to the corresponding chiral hydroxy compounds, an alcohol dehydrogenase from  Lactobacillus minor  and a method for the enantioselective production of (S)-hydroxy compounds from a racemate.

FIELD OF THE INVENTION

[0001] The present invention relates to an enzymic method for theenantioselective reduction of organic keto compounds to give thecorresponding chiral hydroxy compounds, to an alcohol dehydrogenase fromLactobacillus minor and to an enzymic method for enantioselectivelyobtaining (S)-hydroxy compound from a racemate.

BACKGROUND OF THE INVENTION

[0002] Optically active hydroxy compounds are valuable syntheticbuilding blocks for the preparation of a multiplicity ofpharmacologically important compounds. These compounds are oftendifficult to prepare by conventional chemical methods and only rarelyattain the enantiomeric purity required for pharmacologicalapplications. Therefore, biotechnological methods are usually employedin preparing chiral compounds, the stereoselective reaction beingcarried out either by whole microorganisms or using isolated enzymes.

[0003] The use of isolated enzymes has often proved advantageous here,since higher yields and a higher enantiomeric purity are usuallyattainable by using such enzymes.

[0004] Dehydrogenases and in particular alcohol dehydrogenases arevaluable catalysts for obtaining chiral products by stereoselectivereduction of organic keto compounds to the corresponding chiralalcohols. Known enzymes are essentially the corresponding enzymes fromyeast, equine liver or Thermoanaerobium brockii. These enzymes requireNADH (nicotine adenine dinucleotide) or NADPH (nicotine adeninedinucleotide phosphate) as coenzyme. Other examples of known alcoholdehydrogenases are an (S)-specific alcohol dehydrogenase fromRhodococcus erythropolis and an (R)-specific alcohol dehydrogenase fromthe genus Lactobacillus. Both enzyme types have a broad spectrum of ketocompound substrates and have high enantioselectivity. The alcoholdehydrogenases from Lactobacillus kefir (DE 40 14 573) and Lactobacillusbrevis (DE 196 10 984) are particularly suitable for obtaining chiral(R)-alcohols.

[0005] However, the disadvantages of employing alcohol dehydrogenasesare the low enzyme stability and enzyme activity of alcoholdehydrogenases in organic solvents and the frequently only poor watersolubility of the keto compounds to be reduced. Another limiting factorfor employing alcohol dehydrogenases in organic solvents is furthermorethe necessary use of NADP or NAD as cofactor requirement, since thecofactor (NADP, NAD) is water-soluble and is regenerated by economicalmethods.

[0006] It is the object of the invention to improve said disadvantagesby modifying the method conditions. This object is achieved according tothe invention by using a two-phase system comprising an organic solvent,alcohol dehydrogenase, water, cofactor and keto compound.

[0007] The method of the invention has a long stability time due to theenzyme-stabilizing action of the solvent, an enantiomeric purity of morethan 99.9% of the prepared chiral hydroxy compounds and a high yieldbased on the amount of keto compound used.

BRIEF DESCRIPTION OF THE INVENTION

[0008] The method of the invention therefore relates to a method for theenantioselective reduction of a keto compound of the formula I

R¹—C(O)—R²   (I)

[0009] where R¹ and R² are, independently of one another, identical ordifferent and are

[0010] hydrogen,

[0011] 1. —(C₁-C₂₀)-alkyl in which alkyl is straight-chained orbranched,

[0012] 2. —(C₂-C₂₀)-alkenyl in which alkenyl is straight-chained orbranched and, optionally, comprises one, two, three or four doublebonds,

[0013] 4. —(C₂-C₂₀)-alkynyl in which alkynyl is straight-chained orbranched and, optionally, comprises one, two, three or four triplebonds,

[0014] 5. —(C₆-C₁₄)-aryl,

[0015] 6. —(C₁-C₈)-alkyl-(C₆-C₁₄)-aryl or

[0016] 7. R¹ and R² form in combination with the —C(O) radical a—(C₆-C₁₄)-aryl or a —(C₅-C₁₄)-heterocycle,

[0017] where the radicals defined above under 1. to 7. are unsubstitutedor, independently of one another, mono- to trisubstituted by

[0018] a) —OH,

[0019] b) halogen such as fluorine, chlorine, bromine or iodine,

[0020] c) —NO₂,

[0021] d) —C(O)—O—(C₁-C₂₀)-alkyl in which alkyl is linear or branchedand unsubstituted or mono- to trisubstituted by halogen, hydroxyl, aminoor nitro, or

[0022] e) —(C₅-C₁₄)-heterocycle which is unsubstituted or mono- totrisubstituted by halogen, hydroxyl, amino or nitro,

[0023] characterized in that

[0024] a) the compound of the formula I, alcohol dehydrogenase, water,cofactor and an organic solvent having a logP of from 0.5 to 4.0 areincubated

[0025] b) in a two-phase system and

[0026] c) the chiral hydroxy compound is isolated.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Carbon atoms in the ring. Examples of —(C₆-C₁₄)-aryl radicals arephenyl, naphthyl. The term aryl means aromatic carbon radicals havingfrom 6 to 14, for example 1-naphthyl, 2-naphthyl, biphenylyl, forexample 2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl orfluorenyl. Preferred aryl radicals are biphenylyl radicals, naphthylradicals and in particular phenyl radicals. The term “halogen” means anelement of the series fluorine, chlorine, bromine or iodine. The term“—(C₁-C₂₀)-alkyl” means a hydrocarbon radical whose carbon chain isstraight-chained or branched and comprises from 1 to 20 carbon atoms.

[0028] The term “—(C₅-C₁₄)-heterocycle” represents a monocyclic orbicyclic 5-membered to 14-membered heterocyclic ring which is partiallyor completely saturated. Examples of heteroatoms are N, O and S.Examples of the terms—(C₅-C₁₄)-heterocycle are radicals derived frompyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole,thiazole, isothiazole, tetrazole, 1,2,3,5-oxathiadiazole 2-oxides,triazolones, oxadiazolones, isoxazolones, oxadiazolidinediones,triazoles, which are substituted by F, —CN, —CF₃ or—C(O)—O—(C₁-C₄)-alkyl, 3-hydroxypyrro-2,4-diones,5-oxo-1,2,4-thiadiazoles, pyridine, pyrazine, pyrimidine, indole,isoindole, indazole, phthalazine, quinoline, isoquinoline, quinoxaline,quinazoline, cinnoline, carboline and benzo-fused, cyclopenta-,cyclohexa- or cyclohepta-fused derivatives of these heterocycles.Particular preference is given to the radicals 2- or 3-pyrrolyl,phenylpyrrolyl such as 4- or 5-phenyl-2-pyrrolyl, 2-furyl, 2-thienyl,4-imidazolyl, methylimidazolyl, for example 1-methyl-2-, -4- or-5imidazolyl, 1,3-thiazol-2-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-, 3-or 4-pyridyl N-oxide, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl, 2-, 3- or5-indolyl, substituted 2-indolyl, for example 1-methyl-, 5-methyl-,5-methoxy-, 5-benzyloxy-, 5-chloro- or 4,5-dimethyl-2 -indolyl,1-benzyl-2- or -3-indolyl, 4,5,6,7-tetrahydro-2-indolyl,cyclohepta[b]-5-pyrrolyl, 2-, 3- or 4-quinolyl, 1-, 3- or 4-isoquinolyl,1-oxo-1,2-dihydro-3-isoquinolyl, 2-quinoxalinyl, 2-benzofuranyl,2-benzothienyl, 2-benzoxazolyl or benzothiazolyl or dihydropyridinyl,pyrrolidinyl, for example 2- or 3-(N-thiomorpholidinyl), piperazinyl,morpholinyl, thiomorpholinyl, tetrahydrothienyl or benzodioxolanyl.

[0029] Preferred compounds of the formula I are ethyl4-chloro-3-oxo-butanoate, acetophenone, methyl acetoacetate, ethyl2-oxo-4-phenylbutyrate, 2,5-hexanedione, ethyl pyruvate and 2-octanone,preferably ethyl 4-chloro-3-oxobutanoate. The compounds of the formula Iare used in the method of the invention in an amount of from 2% to 30%,based on the total volume, preferably from 10% to 25%, in particularfrom 15% to 22%.

[0030] Preference is given to adding to the water a buffer, for examplepotassium phosphate buffer, Tris/HCl buffer or triethanolamine buffer,having a pH of from 5 to 10, preferably a pH of from 6 to 9. The bufferconcentration is from 10 mM to 150 mM, preferably from 90 mM to 110 mM,in particular 100 mM. The buffer additionally also contains magnesiumions, for example MgCl₂, at a concentration of from 0.2 mM to 10 mM,preferably from 0.5 to 2 mM, in particular 1 mM.

[0031] The temperature is, for example, from about 10° C. to 70° C.,preferably from 30° C. to 60° C.

[0032] The organic solvents that can be used according to the inventionpreferably have a logP of from 0.6 to 2.0, in particular from 0.6 to1.9, particularly preferably from 0.63 to 1.75. Examples of preferredorganic solvents are diethyl ether, tert-butyl methyl ether, diisopropylether, dibutyl ether and ethyl acetate, in particular ethyl acetate.Ethyl acetate may be used, for example, in an amount of from 1% to 90%,based on the total volume of the reaction mixture, preferably from 15%to 60%, in particular from 20% to 50%.

[0033] The ratio of organic solvent to water is from 9 to 1 to 1 to 9,preferably from 1 to 1 to 1 to 3.

[0034] One liquid phase of the two-phase system of the invention iswater and the second liquid phase is the organic solvent. Optionally,there may also still be a solid or another liquid phase produced, forexample, by incompletely dissolved alcohol dehydrogenase or by thecompound of the formula I. Preference, however, is given to two liquidphases without solid phase. The two liquid phases are preferably mixedmechanically so as to generate large surfaces between the two liquidphases.

[0035] The concentration of the NADPH or NADH cofactor, based on theaqueous phase, is from 0.05 mM to 0.25 mM, in particular from 0.06 mM to0.2 mM.

[0036] Preference is given to using in the method of the invention alsoanother stabilizer of alcohol dehydrogenase. Examples of suitablestabilizers are glycerol, sorbitol or dimethyl sulfoxide (DMSO).

[0037] The amount of glycerol is from 5% to 30%, based on the volume ofthe total mixture. Preferred amounts of glycerol are from 10% to 20%, inparticular 20%.

[0038] It is possible to add in the method of the invention additionallyisopropanol in order to regenerate the NADH or NADPH consumed. Forexample, alcohol dehydrogenase converts the isopropanol and NADP toNADPH and acetone.

[0039] The amount of isopropanol used is from 5% to 30%, based on thevolume of the total mixture. Preferred amounts of isopropanol are from10% to 20%, in particular 10%.

[0040] Examples of suitable alcohol dehydrogenases are derived fromyeast, equine liver or Rhodococcus erythropolis, said enzymes requiringNADH as coenzyme, or from Thermoanaerobium brockii, Lactobacillus kefiror Lactobacillus brevis, the latter enzymes requiring NADPH as coenzyme.

[0041] If, for example, an alcohol dehydrogenases of yeast, equineliver, Thermoanaerobium brockii or Rhodococcus erythropolis is used inthe method of the invention, then the corresponding (S)-hydroxy compoundis obtained from the compound of the formula I. If, for example, analcohol dehydrogenases of Lactobacillus kefir or Lactobacillus brevis isused in the method of the invention, then the corresponding (R)-hydroxycompound is obtained from the compound of the formula I.

[0042] The alcohol dehydrogenase may be used in the method of theinvention either in completely purified or in partially purified form orwhen inside cells. The cells used here may be in native, permeabilizedor lysed form.

[0043] The volume activity of the alcohol dehydrogenase used is from 100units/ml (U/ml) to 2000 U/ml, preferably about 800 U/ml, with a proteincontent of about 20 mg/ml to 22 mg/ml. The alcohol dehydrogenasepreferably used has a specific activity of from about 35 to 40 U/mg ofprotein. From 20 000 to 200 000 U, preferably about 100 000 U, ofalcohol dehydrogenase are used per kg of compound of the formula I to beconverted. The enzyme unit 1 U corresponds to the amount of enzymerequired in order to convert 1 μmol of the compound of the formula I perminute (min).

[0044] The method of the invention is carried out, for example, in aclosed reaction vessel made of glass or metal. For this purpose, thecomponents are transferred individually into the reaction vessel andstirred, for example, under a nitrogen or air atmosphere stirring. Thereaction time is from 1 day to 14 days, preferably from 4 to 7 days,depending on the substrate and the compound of the formula I used.

[0045] The reaction mixture is subsequently worked up. For this purpose,the aqueous phase is removed and the ethyl acetate phase is filtered.The aqueous phase can, optionally, be extracted once more and worked upfurther like the ethyl acetate phase. This is followed by evaporatingthe filtered phase under reduced pressure. This results, for example, inthe product ethyl 4-chloro-3(S)-hydroxybutsnoate which has anenantiomeric purity of more than 99.9% and is essentially free of thereactant ethyl 4-chloro-3-oxo-butanoate. After distillation of theproduct, the total yield of the processes is from 82% to 88%, based onthe amount of reactant used.

[0046] Surprisingly, the organic solvents having a logP of from 0 to 4demonstrate a stabilizing action on alcohol dehydrogenase, while theprior art advises against the use of the two-phase systems with organicsolvents (M. R. Kula, U. Kragel; chapter 28, Dehydrogenases in Synthesisof Chiral Compounds; R. N. Patel, Stereoselective Biocatalyses, 2000;Peters J. 9. Dehydrogenases-Characteristics, Design of ReactionConditions, and Application, In: H. J. Rehm, G. Reed Biotechnology, Vol.3, Bioprocessing, V C H Weinheim, 1993; J. Lynda et al., Solventselection strategies for extractive Biocatalysis, Biotechnol. Prog.1991, 7, pages 116-124). The organic phase used in the method of theinvention is ethyl acetate, said organic phase serving on the one handas reservoir for the compound of the formula I but also the reactionproduct, the chiral hydroxy compound, being simultaneously extractedfrom the aqueous phase.

[0047] In contrast to the prior art, the use of organic solvents havinga log-P value of from 0 to 3 results in an additional stabilization ofalcohol dehydrogenase, which increases with time. In the prior art,organic solvents having a log-P value (logarithm of the octanol/waterdistribution coefficient) of from 0 to 2, in particular, have aparticularly destabilizing action on enzymes and are thus hardlyconsidered as organic phase in the two-phase system (K. Faber,Biotransformations in organic chemistry, 3^(rd) edition 1997, SpringerVerlag, chapter 3.to 3.17).

[0048] The invention further relates to Lactobacillus minor alcoholdehydrogenase which has a high temperature optimum. Lactobacillus minoralcohol dehydrogenase has the DNA sequence according to SEQ ID NO: 3 andthe amino acid sequence according to SEQ ID NO: 4 according to theattached sequence protocol. Said Lactobacillus minor alcoholdehydrogenase is R-specific, and it is possible, for example, to obtainfrom a compound of the formula I the corresponding (R)-hydroxy compound.Surprisingly, the enantioselective alcohol dehydrogenase fromLactobacillus minor can be overexpressed in Escherichia coli RB 791,while alcohol dehydrogenases of other species of the genus Lactobacilluswere expressed only to a substantially lower extent. This is all themore surprising, since the wild-type strain of Lactobacillus minoritself expresses only very small amounts of alcohol dehydrogenase whichwas therefore not detectable by common screening methods (whole cellbiotransformation, activity assay). It was therefore very surprisingthat it was possible to clone an R-enantioselective alcoholdehydrogenase from Lactobacillus minor and to overexpress it inEscherichia coli to such an extraordinarily large extent (50% of thecellular proteins of the clone, 20 000 units/g of wet mass).

[0049] The purified enzyme from Lactobacillus minor is stable in a pHrange from about 5.5 to 8.5. The enzyme is stable to about 40° C. andthe pH optimum of the enzymic reaction is in the range from pH 7 to pH7.5. The temperature optimum of the enzymic reaction is about 55° C. Theenzyme has a broad spectrum of substrates.

[0050] The enzyme can be purified to a specific activity of from 35 to40 U/mg of protein by means of hydrophobic interaction chromatography.

[0051] The invention also relates to a method for obtaining alcoholdehydrogenase from Lactobacillus minor. For this purpose, the DNA codingfor Lactobacillus minor alcohol dehydrogenase is expressed in a suitableprokaryotic or eukaryotic microorganism. Lactobacillus minor alcoholdehydrogenase is preferably transformed into and expressed in anEscherichia coli strain, in particular in Escherichia coli RB 791.

[0052]Lactobacillus minor alcohol dehydrogenase can be obtained, forexample, in such a way that the recombinant Escherichia coli cells arecultured, expression of said alcohol dehydrogenase is induced and then,after about 10 to 18 hours (h), the cells are disrupted by ultrasoundtreatment or by means of a French press (Gaullin, Siemens). The cellextract obtained may either be used directly or be purified further. Forthis purpose, the cell extract is centrifuged, for example, and thesupernatant obtained is subjected to a hydrophobic interactionchromatography. Said chromatography is preferably carried out at pH 7.0in an aqueous buffer which also contains magnesium ions.

[0053] The invention further relates to a method for obtaining anenantioselective (S)-hydroxy compound of the formula II

R¹—C(OH)—R²   (II)

[0054] where R¹ and R² are, independently of one another, identical ordifferent and are

[0055] 1. hydrogen,

[0056] 2. —(C₁-C₂₀)-alkyl in which alkyl is straight-chained orbranched,

[0057] 3. —(C₂-C₂₀)-alkenyl in which alkenyl is straight-chained orbranched and, optionally, comprises one, two, three or four doublebonds,

[0058] 4. —(C₂-C₂₀)-alkynyl in which alkynyl is straight-chained orbranched and, optionally, comprises one, two, three or four triplebonds,

[0059] 5. —(C₆-C₁₄)-aryl,

[0060] 6. —(C₁-C₈)-alkyl —(C₆-C₁₄)-aryl or

[0061] 7. R¹ and R² form in combination with the —C(O) radical a—(C₆-C₁₄)-aryl or a —(C₆-C₁₄)-heterocycle,

[0062] where the radicals defined above under 1. to 7. are unsubstitutedor, independently of one another, mono- to trisubstituted by

[0063] a) —OH,

[0064] b) halogen such as fluorine, chlorine, bromine or iodine,

[0065] c) —NO₂,

[0066] d) —C(O)—O—(C₁-C₂₀)-alkyl in which alkyl is linear or branchedand unsubstituted or mono- to tri-substituted by halogen, hydroxyl,amino or nitro, or

[0067] e) —(C₆-C₁₄)-heterocycle which is unsubstituted or mono- totri-substituted by halogen, hydroxyl, amino or nitro,

[0068] characterized in that

[0069] a) a racemic mixture comprising the compound of the formula II,the alcohol dehydrogenase of the invention, water, cofactor and anorganic solvent an organic solvent having a logP of from 0.5 to 4.0, forexample from the series diethyl ether, tert-butyl methyl ether,diisopropyl ether or ethyl acetate, is incubated

[0070] b) in a two-phase system and

[0071] c) the enantiomerically pure (S)-hydroxy compound formed isisolated.

[0072] The reaction conditions are essentially the same as in theabovementioned method for the enantiospecific reduction of the ketocompound of the formula I. However, instead of enantioselectivelyreducing the keto compound of the formula I, the method comprisesoxidizing the corresponding (R)-hydroxy compound of the formula II tothe corresponding keto compound. Furthermore, the method uses acetonerather than isopropanol for regenerating NADP. For example, the alcoholdehydrogenase of the invention converts acetone and NADPH to NADP andisopropanol. The amount of acetone used is from 5% to 30%, based on thevolume of the total mixture. Preferred amounts of acetone are from 10%to 20%, in particular 10%.

[0073] The alcohol dehydrogenase of the invention may be present forpreparation of the compound of the formula II in either completely orpartially purified form or may also be used in said method when insidecells. Said cells may be present here in a native, permeabilized orlysed form.

[0074] The invention also relates to a recombinant Escherichia coliclone, RB 791, which expresses Lactobacillus minor alcohol dehydrogenaseand which was deposited under the conditions of the Budapest Treaty withthe Deutsche Sammlung für Mikroorganismen und Zellkulturen, MascheroderWeg 1b, 38124 Brunswick on Mar. 26, 2001 under the number DSM 14196.

[0075] The invention is illustrated by the following examples:

EXAMPLE 1 Screening for R-alcohol Dehydrogenases in Strains of the GenusLactobacillus by Means of Whole Cell Biotransformation

[0076] Various Lactobacillus strains were cultured for screening in thefollowing medium (numbers in each case in g/l): glucose (20), yeastextract (5), meat extract (10), diammonium hydrogen citrate (2), sodiumacetate (5), magnesium sulfate (0.2), manganese sulfate (0.05),dipotassium hydrogen phosphate (2).

[0077] The medium was sterilized at 121° C. and the strains of the genusLactobacillus (abbreviated to L. hereinbelow) were cultured withoutfurther pH regulation or addition of oxygen. The cells weresubesequently removed by centrifugation, and in each case 4 g of cellswere resuspended for whole cell biotransformation in a final volume of10 ml of potassium phosphate buffer (KPi buffer) (50 mM, pH=7.0). Afteraddition of in each case 0.1 g of glucose, the cells were shaken at 30°C. for 15 min. Ethyl 4-chloro-3-oxo-butanoate was added at a finalconcentration of 40 mM to the cell suspension, and the medium wasanalyzed by gas chromatography in each case after 10 min and 120 min.For this purpose, the cells were removed by centrifugation, thesupernatant was filtered and diluted in chloroform to a finalconcentration of 10-15 μg/ml of ethyl 4-chloro-3-oxobutanoate.

[0078] The various Lactobacillus strains were used to convert ethyl4-chloro-3-oxobutanoate, used as substrate, with the followingenantiomeric purity to ethyl(S)-4-chloro-3-hydroxybutyrate.

[0079] The enantiomeric excess is calculated as follows:

ee(%)=((R-alcohol−S-alcohol)/(R-alcohol+S-alcohol))×100. TABLE 1 ee ofethyl 4-chloro-3- Lactobacillus strain (S)-hydroxybutanoate in % L.reuteri 34.6 L. kandleri 90 L. collinoides 71.3 L. bifermentans 53.6 L.oris 63.4 L. brevis 74 L. halotolerans 67.2 L. minor 18.6 L.parabuchneri 78.5 L. kefir 87.8 L. fructosus 28.9

EXAMPLE 2 Obtaining Recombinant R-Specific Alcohol Dehydrogenases

[0080] A.) Preparation of Genomic DNA from Strains of the GenusLactobacillus

[0081] The cell pellet of approximately 2 ml of culture liquid of thegenus Lactobacillus was resuspended in 300 μl of TE buffer (containing10 mM Tris/HCl, pH=8.1 mM EDTA), admixed with 20 mg/ml lysozyme andincubated at 37° C. for 10 min. This was followed by adding 100 μl ofsodium dodecylsulfate (SDS) (10%), 100 μl of sodium perchlorate (5M) and500 μl of chloroform/isoamyl alcohol (24:1). After shaking vigorously,the protein was removed by centrifugation and the aqueous phasetransferred to a new Eppendorf vessel and this was followed by adding800 μl of ethanol (EtOH) (96%). The Eppendorf vessel was invertedseveral times and the precipitated chromosomal DNA then transferred to anew Eppendorf vessel and washed with 200 μl of EtOH. The DNA was againtransferred to a new Eppendorf vessel, dried under reduced pressure anddissolved in 100 μl of TE buffer.

[0082] B.) Oligonucleotides as 5′ and 3′ primers for PCR (PolymeraseChain Reaction)

[0083] The primers used for the PCR were derived from the knownN-terminal and C-terminal sequence of L. kefir alcohol dehydrogenase,with known preferences for particular codons in lactobacilli being takeninto account. Thus, the codon ATG (Met) as start codon was put in frontof each 5′ primer, and furthermore the cleavage site for the restrictionenzyme Bam HI (GGATCC) was located upstream of said start codon on the5′ primer, in order to enable subsequent cloning into the expressionsector. The stop codon (TAG) and the cleavage site for Hind III (AAGCTT)were placed downstream of the 3′ primer. The primer constructs arelisted below:

[0084] N=A, C or G; Y=T or C; R=A or G 5′ primer5′GCGGATCCATGACNGAYCGNTTRAARGGNAARGTN (SEQ ID NO:1) GC3′ 3′ primer5′GGGAAGCTTCTAYTGNGCNGTRTANCCNCCRTCNA (SEQ ID NO:2) C3′

[0085] The primers were prepared according to known methods.

[0086] C.) PCR (Polymerase Chain Reaction) with Genomic DNA from Strainsof the Genus Lactobacillus

[0087] PCR Mixture (100 μl): Amount used per reaction ConcentrationdNTP's  8 μl per NTP 2.5 nmol/μl Oligos per oligo 10 μl: 20 μl 2 pmol/μlChromosomal DNA  3 μl ca. 1 μg/μl 10 × buffer 10 μl (Promega) Taqpolymerase  1 μl 2 U/μl (Promega) H₂O 58 μl

[0088] Cycle:

[0089] 95° C. for 2 min, followed by maintaining 80° C.

[0090] hot start, followed by

[0091] 95° C. for 30 sec, followed by

[0092] 40° C. for 1 min 30×

[0093] followed by in each case 30 times 95° C. for 30 s and 40° C. for1 min, then

[0094] 72° C. for 2.5 min, followed by

[0095] 72° C. for 2.5 min followed by

[0096] maintaining 10° C.

[0097] For analysis, 10 μl of the mixture were applied to a 1% strengthagarose gel and electrophoretically fractionated at a constant 100 V.The PCR revealed distinct amplification of a DNA piece of approximately750 bp.

[0098] D.) Isolation of PCR Fragments from the Gel

[0099] In order to obtain the PCR fragment, the entire PCR mixture wasapplied to a 1% strength agarose gel and electrophoreticallyfractionated at a constant 100 V. For this purpose, the gel was dividedinto two lanes, one containing the complete PCR mixture and the otherone containing only a sample of 5 μl, so that the PCR fragment wasexcised from the gel by staining with ethidium bromide only the lanewith the sample for orientation purposes, in order to rule out damagedue to ethidium bromide and UV light of the PCR fragment to be isolated.

[0100] Isolation from the gel was carried out using the QIAquick GelExtraction Kit from Qiagen, Hilden, Germany.

[0101] A total concentration of 20 ng/μl DNA was determined.

[0102] E.) Ligation

[0103] To prepare the ligation, the purified PCR fragment and thecloning vector used, pQE30 or pQE70, both from Quiagen, were cleavedwith Bam HI and Hind III (4 μl of DNA=200 ng of DNA, 1 μl of 10× buffer,1 μl of enzyme, BSA and H₂O (Biolabs, New England)).

[0104] The cleaved plasmid was then purified again by means of theQIAquick Gel Extraction Kit, taken up in water, dephosphorylated bymeans of alkaline phosphatase (USB, Amersham Life Science).

[0105] For purification, the appropriate reaction mixtures were againapplied to a 1% strength agarose gel, and thus the digested amplicon andthe plasmid were isolated from the gel, as described under D.). Theconcentration of plasmid and amplicon after purification wasapproximately 20 ng/μl.

[0106] For ligation, 3 μl of pQE30 or pQE70 (60 ng), 2.5 μl of amplicon(50 ng), 2 μl of ligase buffer (Boehringer; Mannheim), 1.5 μl of H₂O and1 μl of T4 ligase (Boehringer; Mannheim) were used. The mixture wasincubated at 16° C. overnight.

[0107] Subsequently, 40 μl of electrocompetent Escherichia coli RB791cells were transformed with 1.5 μl of ligation mixture byelectroporation. The cells were introduced to 500 μl of SOC medium,incubated at 37° C. for 45 min and then in each case 250 μl were platedout on LB_(amp) agar plates. The SOC medium contains per liter of water20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, 10 ml of 1 MMgSO₄ and 10 ml of 1 M MgCl₂. LB_(amp)agar plates contain per liter ofwater 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 20 g ofagar, pH 7.0, and 50 mg of ampicillin.

[0108] Grown colonies were removed and cultured in 4 ml of liquidculture (LB_(amp) medium) at 37° C. overnight. In each case 2 ml of thiscell suspension were used for plasmid preparation (according to theQuiagen miniprep protocol (Quiagen, Hilden, Germany)).

[0109] The plasmid was prepared starting with a Bam HI and Hind IIIrestriction digest. The complete digest was applied to a 1% strengthagarose gel and electrophoretically fractionated at 100 V (detection ofthe 750 kp insert), followed by using the plasmids for sequencing,optionally.

[0110] Clones having the 750 kp insert were then plated out on LB_(amp)agar plates.

[0111] F.) Sequencing of Plasmids

[0112] Sequencing was carried out by means of SequiThermEXCEL IILong-Read DNA Sequencing Kit (Biozym, Oldendorf, Germany) on an Li-Corsequencer (MWG Biotech, Ebersberg, Germany), according to themanufacturer's instructions. The primers used were the standardsequencing primers for pQE vectors.

[0113] G.) Screening of Clones with Respect to Soluble R-ADH Expression

[0114] Clones having inserts of 750 kp were studied with regard toenzymic activity and stereoselectivity. For this purpose, the cloneswere removed from the LB_(amp) agar plates and cultured in 20 ml ofliquid cultures (LB_(amp) medium) at 25° C. and then, at a cell density(OD₅₀₀) of 0.5, induced with 1 mM isopropyl-β-D-thiogalactopyranoside(IPTG). After 18 h, the cells were removed by centrifugation and in eachcase 40 mg of cells were taken up in 350 μl of Kpi buffer (50 mM, pH=7,1 mM MgCl₂). The enzyme was liberated from the cells by wet grindingwith the aid of glass beads (0.5 g, 0.3 mm). In addition, the cells weredisrupted by means of a Retsch mill at 4° C. for 20 minutes.

[0115] The enzyme assay contained 870 μl of triethanolamine buffer (100mM, pH=7.0, 1 mM MgCl₂), 100 μl of a 100 mM solution of ethyl4-chloro-acetoacetate, 10 μl of NADPH (final concentration 0.19 mM) and20 μl of enzyme solution.

[0116] Enzyme unit definition: 1 U corresponds to the amount of enzymerequired for converting 1 μmol of substrate (ethyl4-chloro-3-oxobutanoate) per 1 min.

[0117] Stereoselectivity was detected by incubating 480 μl oftriethanolamine buffer (100 mM, pH=7.0, 1 mM MgCl₂) with 1.0 mM ethyl4-chloro-3-oxobutanoate, 1.9 mM NADPH (in each case final concentration)and 20 μl of enzyme solution. After incubating for 15 min, the reactionmixture was filtered and diluted 1:10 in chloroform, and a sample wasanalyzed by means of GC-MS.

[0118] Conditions of Gas Chromatography (GC):

[0119] Chiral column: Lipodex E, ID=0.25 mm, 1=25 m (Macherey-Nagel)

[0120] 1. 2 min 60° C.

[0121] 2. in 28 min from 60° C. to 130° C. with a rate of 2.5° C. perminute

[0122] 3. 15 min at 130° C.

[0123] An (R)-specific alcohol dehydrogenase was able to be cloned andactively overexpressed from the following Lactobacillus strains:Activity Clone in U/g of Strain Plasmid number cells* ee in % L.parabuchneri pQE30 12 450 >99.9 L. parabuchneri pQE30 14 170 >99.9 L.kandleri pQE30 11 280 >99.9 L. kandleri pQE70 17 710 >99.9 L. minorpQE30 2 2 830 >99.9 L. minor pQE70 3 680 >99.9 L. minor pQE70 4 700>99.9

[0124] H.) Enzyme Obtainment and Purification

[0125] The enzyme was obtained by culturing the strain with the highestenzymic activity in a fermenter (fed batch, 10 l) and inducing it at 40OD₅₀₀ with 1 mM IPTG. After 18 h, the cells were harvested, taking up300 g of cells in 3 l of Kpi buffer (50 mM, pH=7, 1 mM MgCl₂) anddisrupted subsequently by means of a French press (Gaullin, Siemens).The supernatant obtained after centrifugation is referred to as crudeextract hereinbelow and had a volume activity of approximately 2000 U/ml(20 000 U/g of wet mass).

[0126] To characterize the enzyme, a portion of the enzyme obtained waspurified by means of hydrophobic interaction chromatography onQ-Sepharose ff (fast flow). For this purpose, the column used wasequilibrated with 50 mM Kpi buffer pH=7.0, 1 mM MgCl₂. After applicationof the crude extract to the column and brief washing with equilibrationbuffer, the enzyme was eluted with an increasing linear salt gradient(0-1M NaCl, 1 ml/min) at a salt concentration of about 0.3 M NaCl.Combining the enzyme-containing fractions resulted in approximately 25ml of purified enzyme with a volume activity of about 800 U/ml and aprotein content of from 20 to 22 mg/ml. The enzyme purified in this waythus has a specific activity of approximately 35 to 40 U/mg of protein.

[0127] All enzymic activities were determined at 25° C. The enzymeactivity was calculated as follows:

[0128] Calculation: 1 unit=conversion of 1 μmol of substrate

[0129] per min

[0130] Lambert-Beer law

[0131] NADPH decrease was monitored at 340 nm (see enzymic assaymixture)=ΔE/min

[0132] N=enzyme dilution factor

[0133] V=enzyme volume in ml (0.01)

[0134] V_(cuvette)=cuvette volume=1 ml

[0135] d=cuvette light path=1 cm

[0136] e_(NADPH)=NADPH extinction coefficient=6.22 [mM⁻¹*cm⁻¹]

Activity=(ΔE/min*N*V _(cuvette))/(e _(NADPH) *V*d)

[0137] Protein determination was carried out according to Bradford(Bio-Rad Laboratories GmbH, Protein Assay).

EXAMPLE 3 Enzyme-Catalyzed Preparation ofethyl(S)-4-chloro-3-hydroxybutyrate

[0138] A.) On a 5-Liter Scale

[0139] The alcohol dehydrogenase crude extract obtained in Example 2 andthe coenzyme NADP were employed in the enzyme-catalyzed synthesis ofethyl(S)-4-chloro-3-hydroxybutyrate from ethyl 4-chloro-3-oxobutanoate.The oxidized coenzyme was continuously regenerated due to theconcomitant presence of isopropanol so that the reaction requires onlycatalytic amounts of coenzyme.

[0140] The mixture contained:

[0141] 2 l of triethanolamine buffer 100 mM pH=7.0, 1 mM

[0142] MgCl₂, 10 % glycerol,

[0143] 400 mg of NADP,

[0144] 600 ml of isopropanol,

[0145] 800 ml of ethyl acetate,

[0146] 600 ml of ethyl 4-chloro-3-oxobutanoate and approximately 100 000units of alcohol dehydrogenase.

[0147] After 3 days of stirring at room temperature, complete conversionof ethyl 4-chloro-3-oxo-butanoate to ethyl(S)-4-chloro-3-hydroxybutyratewith enantiomeric purity of more than 99.9% was detected by gaschromatography.

[0148] After removing the aqueous phase, evaporating the solvent and,optionally, distillation, purified ethyl(S)-4-chloro-3-hydroxybutyrateis obtained with an enantiomeric purity of more than 99.9%.

[0149] B.) On a 50 l Scale

[0150] The reaction mixture for converting 10 l of ethyl4-chloro-3-oxo-butanoate is composed as follows:

[0151] 18 l of triethanolamine buffer 100 mM pH=7.0, 1 mM

[0152] MgCl₂, 10% glycerol,

[0153] 4 g of NADP,

[0154] 10 l of isopropanol,

[0155] 10 l of ethyl acetate,

[0156] 10 l of ethyl 4-chloro-3-oxo-butanoate and approximately 2million units of alcohol dehydrogenase (1.25 l of crude extract).

[0157] After 7 days of stirring at room temperature, complete conversionof ethyl 4-chloro-3-oxo-butanoate to ethyl(S)-4-chloro-3-hydroxybutyratewith enantiomeric purity of more than 99.9% was detected by gaschromatography.

EXAMPLE 4 Biochemical Characterization of Cloned Alcohol Dehydrogenasefrom Lactobacillus Minor

[0158] A.) pH Stability

[0159] The activity of the enzyme as a function of storage in bufferswith different pH values in the range from pH 4 to 11 was studied. Forthis purpose, various buffers (50 mM) in the range from pH 4 to 11 wereprepared and the enzyme purified in Example 2 was diluted 1:100 thereinand incubated for 30 min. All buffers contained 1 mM MgCl₂. 10 μl ofthis were then used in the normal enzyme assay (triethanolamine buffer100 mM pH=7.0, 1 mM MgCl₂, 10 mM ethyl 4-chloro-3-oxo-butanoate and 0.19mM NADPH). The reaction was monitored at 30° C. and 340 nm for 1 min.

[0160] The starting value here is the measured value obtainedimmediately after diluting the enzyme in triethanolamine buffer 50 mMpH=7.0. Under predefined conditions, this value corresponded to a changein extinction of 0.20/min and was set as 100% value, with all subsequentmeasured values being related to this value. TABLE 2 Activity in %Activity in % pH Buffer system (n = 2) Buffer system (n = 2) 4 sodiumacetate/ 87.5 ± 6.5 acetic acid 4.5 sodium acetate/ 94.5 ± 3.0 aceticacid 5 sodium acetate/ 94.5 ± 1.5 MES/NaOH 55 ± 5 acetic acid 5.5KH₂PO₄/K₂PO₄ 96 ± 3 MES/NaOH 77.1 ± 2.1 6 KH₂PO₄/K₂PO₄ 100 ± 0 triethanol- 100 ± 0  amine/NaOH 6.5 KH₂PO₄/K₂PO₄ 97.5 ± 2.5 triethanol-100 ± 0  amine/NaOH 7 KH₂PO₄/K₂PO₄ 100 ± 0  triethanol- 97.9 ± 2.1amine/NaOH 7.5 KH₂PO₄/K₂PO₄ 97.5 ± 7.5 tris/HCl 94.6 ± 1.3 8KH₂PO₄/K₂PO₄ 93.0 ± 3.0 tris/HCl 89.2 ± 0   8.5 KH₂PO₄/K₂PO₄ 102.5 ±2.5  tris/HCl   60 ± 4.2 9 glycine/NaOH 76.5 ± 1.5 tris/HCl 63.1 ± 4.89.5 glycine/NaOH 52.5 ± 7.5 10 glycine/NaOH 52.5 ± 7.5 11 glycine/NaOH0.0 ± 0 

[0161] Table 2 indicates that the enzyme has good pH stability, inparticular in the acidic range, the enzyme stability appearing to be afunction not only of the pH but also of the buffer system used. Whenusing, for example, TRIS and MES buffers, the enzyme is found to beinactivated more strongly than in the KPi buffer with the same pH. Therewas no significant inactivation in the KPi buffer in the pH range from5.5 to 8.5.

[0162] B.) Temperature Stability

[0163] The temperature stability for the range from 25° C. to 50° C. wasdetermined similarly to the manner described in A.). For this purpose,in each case a 1:100 dilution of the purified enzyme was incubated atthe particular temperature for 30 min and then measured at 30° C. usingthe above assay procedure. Here too, the starting value used was themeasured value obtained immediately after diluting the enzyme intriethanolamine buffer 50 mM pH=7.0. This value was also set here as100% value. L. minor alcohol dehydrogenase is stable up to a temperatureof 40° C. Thereafter, the activity rapidly declines. TABLE 3 Activity inActivity in Temperature % (n = 4) Temperature % (n = 4) 25  101 ± 3.2 4033.4 ± 3.8  30 81.2 ± 5.8 42 0 ± 0 35 67.0 ± 1.6 45 0 ± 0 37 20.2 ± 2.450 0 ± 0

[0164] C.) pH Optimum

[0165] The pH optimum was determined by determining the enzymic reactionin the relevant buffer listed in Table 3. As in the standard assay, theconcentration of ethyl 4-chloro-3-oxo-butanoate and of NADPH was 10 mMand 0.19 mM, respectively. The reaction was determined at 30° C. Theenzyme of the invention was found to have a pH optimum between 7 and7.5. TABLE 4 Activity in U/ml of undiluted pH Buffer system enzyme 4sodium acetate/acetic acid 85 4.5 sodium acetate/acetic acid 132 5MES/NaOH 218 5.5 MES/NaOH 240 6 triethanolamine/NaOH 381 6.5triethanolamine/NaOH 349 7 triethanolamine/NaOH 510 7.5 tris/HCl 707 8tris/HCl 585 8.5 tris/HCl 486 9 tris/HCl 488 10 glycine/NaOH 131 11glycine/NaOH 0

[0166] D.) Temperature Optimum

[0167] The optimal assay temperature was determined by measuring theenzyme activity from 25° C. to 60° C. The assay mixture corresponded tothe standard concentration of ethyl 4-chloro-3-oxo-butanoate and NADPH.As Table 5 demonstrates, the optimal assay temperature of the enzyme is55° C., with the activity declining rapidly thereafter. TABLE 5 Activityin U/ml of undiluted Temperature enzyme 25 540 30 1235 35 1968 40 162145 2469 50 2469 55 2855 60 0

[0168] E.) Spectrum of Substrates

[0169] Furthermore, substrates other than ethyl 4-chloro-3-oxo-butanoatewere also used in the enzymic assay mixture. For this purpose, thefollowing assay mixture was used:

[0170] 970 μl of triethanolamine buffer (100 mM, pH=7.0, 1 mM

[0171] MgCl₂ containing 10 mM keto compound)

[0172] 20 μl of NADPH (0.19 mM in assay mixture)

[0173] 10 μl of enzyme (1:100)

[0174] The activity determined with ethyl 4-chloro-3-oxo-butanoate wasset to 100% and the enzyme activities of the other substrates wererelated to this value. TABLE 6 Substrate Activity in % (n = 2) Ethyl4-chloro-3-oxo-butanoate 100 Ethyl pyruvate 192.3 ± 11.5 2-Octanone 90.8± 1.2 Methyl acetoacetate  120 ± 7.7 Ethyl 2-oxo-4-phenylbutyrate 62.7 ±4.8

[0175] F.) Enzyme Stability in Organic Solvents

[0176] The stability of the enzyme when contacted with organic solventswas studied by diluting L. minor alcohol dehydrogenase 1:100 in thesolvent mixtures listed, followed by incubation at room temperature (fororganic solvents not miscible with water, the dilution refers to theaqueous phase). Continuous mixing of both phases was ensured (shaker,200 rpm). 10 μl of the enzyme solution were then used in the standardassay mixture. Here too, the starting value was set to 100% afterdilution in the buffer (triethanolamine buffer 100 mM, pH=7.0, 1 mMMgCl₂), with all other values being related to this value. TABLE 7 A.)Water-miscible solvents: Solvent logP t = 2 h t = 8 h t = 24 h t = 48 hBuffer 86 70 3 0 10% isopropanol 0.28 32 34 16 0 20% isopropanol 0.28 1617 7 0 10% DMSO −1.3 73 54 60 40 20% DMSO −1.3 73 54 57 40 1M sorbitol93 74 60 6 10% glycerol −3 120 64 62 28 20% glycerol −3 120 100 100 104

[0177] As Table 7A demonstrates, glycerol, DMSO and sorbitol have anactivating and, respectively, stabilizing action on the alcoholdehydrogenase used. In contrast, the isopropanol to be used in theprocess has an inactivating action. B.) Solvent not miscible with waterSolvent logP t = 2 h t = 8 h t = 24 h t = 48 h Buffer 86 70 3 0 20%ethyl acetate 0.68 87 50 10 8 20% diethyl ether 0.85 53 42 37 23 20%tert-butyl methyl ether 1.21 67 51 38 24 20 diisopropyl ether 1.55 10057 41 29 20% dibutyl ether 2.9 92 71 23 6 20% pentane 3 74 55 7 6 20%hexane 3.5 80 39 2 5 20% heptane 4 51 49 7 6 20% octane 4.5 87 47 2 1

[0178] As Table 7B demonstrates, the alcohol dehydrogenase studiedexhibits considerable stability in a large number of organic solvents.Surprisingly, solvents having logP values between 0 and 3 inhibit thealcohol dehydrogenase studied no more than those having logP valuesbetween 3 and 4.5, in particular with regard to longer incubation times(24 h and 48 h) solvents having logP values betwen 0 and 3 stabilize theADH studied, compared to the corresponding values in the buffer. Thealiphatic solvents studied, pentane, hexane, heptane and octane, do notexhibit this stabilizing action with long-term incubation.

[0179] The logP value of a component X is the logarithm of the

[0180] distribution coefficient of X in the octanol/water two-phasesystem (50/50)

[0181] P=concentration of X in octanol phase/concentration of X inaqueous phase

[0182] G. Enzyme Stability Under Process Conditions

[0183] The stability of the enzyme under process conditions was studiedby diluting L. minor alcohol dehydrogenase 1:100 with the solventmixtures used in the two-phase system, followed by incubation at roomtemperature. 10 μl of the enzyme solution were then used in the standardassay mixture.

[0184] Table 8 depicts the enzyme activities as a % of the startingvalue. TABLE 8 6 h 20 h 46 h 60 h 84 h Triethanolamine 100 75 0 0 0buffer, 100 mM, 1 mM MgCl₂ Mixture B 100 85 80 60 55 Mixture C 110 95 9585 80 Mixture D 100 65 55 50 50

[0185] It was found that recombinant L. minor alcohol dehydrogenase isstable and active in the combination of solvents used in the two-phasesystem for several days.

1 4 1 795 DNA Artificial Sequence Description of Artificial Sequence DNAprimer 1 atgagaggat cgcatcacca tcaccatcac ggatccatga ccgatcggttgaaggggaaa 60 gtagcaattg taactggcgg taccttggga attggcttgg caatcgctgataagtttgtt 120 gaagaaggcg caaaggttgt tattaccggc cgtcacgctg atgtaggtgaaaaagctgcc 180 agatcaatcg gcggcacaga cgttatccgt tttgtccaac acgatgcttctgatgaaacc 240 ggctggacta agttgtttga tacgactgaa gaagcatttg gcccagttaccacggttgtc 300 aacaatgccg gaattgcggt cagcaagagt gttgaagata ccacaactgaagaatggcgc 360 aagctgctct cagttaactt ggatggtgtc ttcttcggta cccgtcttggaatccaacgt 420 atgaagaata aaggactcgg agcatcaatc atcaatatgt catctatcgaaggttttgtt 480 ggtgatccag ctctgggtgc atacaacgct tcaaaaggtg ctgtcagaattatgtctaaa 540 tcagctgcct tggattgcgc tttgaaggac tacgatgttc gggttaacactgttcatcca 600 ggttatatca agacaccatt ggttgacgat cttgaagggg cagaagaaatgatgtcacag 660 cggaccaaga caccaatggg tcatatcggt gaacctaacg atatcgcttggatctgtgtt 720 tacctggcat ctgacgaatc taaatttgcc actggtgcag aattcgttgtcgacggaggg 780 tacaccgccc aatag 795 2 37 DNA Artificial SequenceDescription of Artificial Sequence DNA primer 2 gcggatccat gacngaycgnttraarggna argtngc 37 3 36 DNA Artificial Sequence Description ofArtificial Sequence DNA primer 3 gggaagcttc taytgngcng trtanccncc rtcnac36 4 264 PRT Lactobacillus sp. 4 Met Arg Gly Ser His His His His His HisGly Ser Met Thr Asp Arg 1 5 10 15 Leu Lys Gly Lys Val Ala Ile Val ThrGly Gly Thr Leu Gly Ile Gly 20 25 30 Leu Ala Ile Ala Asp Lys Phe Val GluGlu Gly Ala Lys Val Val Ile 35 40 45 Thr Gly Arg His Ala Asp Val Gly GluLys Ala Ala Arg Ser Ile Gly 50 55 60 Gly Thr Asp Val Ile Arg Phe Val GlnHis Asp Ala Ser Asp Glu Thr 65 70 75 80 Gly Trp Thr Lys Leu Phe Asp ThrThr Glu Glu Ala Phe Gly Pro Val 85 90 95 Thr Thr Val Val Asn Asn Ala GlyIle Ala Val Ser Lys Ser Val Glu 100 105 110 Asp Thr Thr Thr Glu Glu TrpArg Lys Leu Leu Ser Val Asn Leu Asp 115 120 125 Gly Val Phe Phe Gly ThrArg Leu Gly Ile Gln Arg Met Lys Asn Lys 130 135 140 Gly Leu Gly Ala SerIle Ile Asn Met Ser Ser Ile Glu Gly Glu Val 145 150 155 160 Gly Asp ProAla Leu Gly Ala Tyr Asn Ala Ser Lys Gly Ala Val Arg 165 170 175 Ile MetSer Lys Ser Ala Ala Leu Asp Cys Ala Leu Lys Asp Tyr Asp 180 185 190 ValArg Val Asn Thr Val His Pro Gly Tyr Ile Lys Thr Pro Leu Val 195 200 205Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln Arg Thr Lys Thr 210 215220 Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala Trp Ile Cys Val 225230 235 240 Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly Ala Glu PheVal 245 250 255 Val Asp Gly Gly Tyr Thr Ala Gln 260

1. A method for the enantioselective reduction of a keto compound of theformula I R¹—C(O)—R²   (I) where R¹ and R² are, independently of oneanother, identical or different and are
 1. hydrogen,
 2. —(C₁-C₂₀)-alkylin which alkyl is straight-chained or branched,
 3. —(C₂-C₂₀)-alkenyl inwhich alkenyl is straight-chained or branched and, optionally, comprisesone, two, three or four double bonds,
 4. —(C₂-C₂₀)-alkynyl in whichalkynyl is straight-chained or branched and, optionally, comprises one,two, three or four triple bonds,
 5. —(C₆-C₁₄)-aryl,
 6. —(C₁-C₈)-alkyl—(C₆-C₁₄)-aryl or
 7. R¹ and R² form in combination with the —C(O)radical a —(C₆-C₁₄)-aryl or a —(C₅-C₁₄)-hetercycle, where the radicalsdefined above under
 1. to
 7. are unsubstituted or, independently of oneanother, mono- to trisubstituted by a) —OH, b) halogen such as fluorine,chlorine, bromine or iodine, c) —NO₂, d) —C(O)—O—(C₁-C₂₀)-alkyl in whichalkyl is linear or branched and unsubstituted or mono- to trisubstitutedby halogen, hydroxyl, amino or nitro, or e) —(C₅-C₁₄)-heterocycle whichis unsubstituted or mono- to trisubstituted by halogen, hydroxyl, aminoor nitro, said method comprising a) a compound of the formula I with aproportion of equal to/greater than 5% to 30%, based on the total volumeof the reaction mixture, alcohol dehydrogenase, water, cofactor NADPH orNADH and an organic solvent immiscible with water and having a logP offrom 0.5 to 4.0; b) are incubated in a two-phase system of water andorganic solvent immiscible with water; c) the oxidized cofactor producedby said alcohol dehydrogenase is steadily regenerated, and d) the chiralhydroxy compound is isolated.
 2. The method as claimed in claim 1,wherein a compound of the formula I of the series ethyl4-chloro-3-oxobutanoate, acetophenone, methyl acetoacetate, ethyl2-oxo-4-phenylbutyrate, 2,5-hexanedione, ethyl pyruvate or 2-octanone isused.
 3. The method as claimed in claim 1, wherein an organic solventhaving a logP of from 0.6 to 3.0, is used.
 4. The method as claimed inclaim 3, wherein an organic solvent having a logP of from 0.63 to 1.75is used.
 5. The method as claimed in claim 1, wherein the organicsolvent used is diethyl ether, tert-butyl methyl ether, diisopropylether or ethyl acetate.
 6. The method as claimed in claim 1, wherein analcohol dehydrogenases from yeast, equine liver, Thermoanaerobiumbrockii, Rhodococcus erythropolis, Lactobacillus kefir, Lactobacillusbrevis, Lactobacillus minor or an alcohol dehydrogenase having the aminoacid sequence according to SEQ ID NO: 4 is used.
 7. The method asclaimed in claim 1, wherein a buffer selected from potassium phosphate,Tris/HCl or triethanolamine buffer, having a pH of from 5 to 10, isadded.
 8. The method as claimed in claim 7, wherein magnesium ions at aconcentration of from 0.2 mM to 10 mM are added to the buffer.
 9. Themethod as claimed in claim 1, wherein the cofactor added is NADPH orNADH in an amount of from 0.01 mM to 0.25 mM, based on the aqueousphase.
 10. The method as claimed in claim 1, wherein glycerol, sorbitolor dimethyl sulfoxide is added as stabilizer for alcohol dehydrogenase.11. The method as claimed in claim 1, wherein isopropanol is added. 12.The method as claimed in claim 1, wherein the compounds of the formula Iare used in an amount of from 5% to 30% based on the total volume. 13.The method as claimed in claim 1, wherein the reaction is carried out ata temperature of from about 10° C. to 70° C.
 14. The method as claimedin claim 1, wherein the organic solvent are used in an amount of from 1%to 90%, based on the total volume of the reaction mixture.
 15. Themethod as claimed in claim 1, wherein the ratio of organic solvent towater is from 9 to 1 to 1 to
 9. 16. The method as claimed in claim 10,wherein the stabilizer is used in an amount of from 5% to 30%, based onthe volume of the total reaction mixture.
 17. The method as claimed inclaim 11, wherein isopropanol is used in an amount of from 5% to 30%,based on the volume of the total reaction mixture.
 18. The method asclaimed in claim 6, wherein the alcohol dehydrogenase is used in anamount of from 20 000 U to 200 000 U per kg of compound of the formula Ito be reacted.
 19. The method according to claim 18, comprising aLactobacillus minor alcohol dehydrogenase having the amino acid sequenceaccording to SEQ ID NO:
 4. 20. The method according to claim 18,comprising a Lactobacillus minor alcohol dehydrogenase having the aminoacid sequence according to SEQ ID NO:
 3. 21. A method for obtaining thealcohol dehydrogenase from Lactobacillus minor as claimed in claim 19,wherein the DNA coding for Lactobacillus minor alcohol dehydrogenase isexpressed in a suitable prokaryotic or eukaryotic microorganism, inparticular in cells of Escherichia coli cell deposited under DSM 14196,and, optionally, said alcohol dehydrogenase is purified.
 22. A methodfor obtaining an enantioselective (S)-hydroxy compound of the formula IIR¹—C(OH)—R²   (II) where R¹ and R² are, independently of one another,identical or different and are
 1. hydrogen,
 2. —(C₁-C₂₀)-alkyl in whichalkyl is straight-chained or branched,
 3. —(C₂-C₂₀)-alkenyl in whichalkenyl is straight-chained or branched and, optionally, comprises one,two, three or four double bonds,
 4. —(C₂-C₂₀)-alkynyl in which alkynylis straight-chained or branched and, optionally, comprises one, two,three or four triple bonds,
 5. —(C₆-C₁₄)-aryl, 6.—(C₆-C₈)-alkyl—(C₆-C₁₄)-aryl or
 7. R¹ and R² form in combination withthe —C(O) radical a —(C₆-C₁₄)-aryl or a —(C₆-C₁₄)-heterocycle, where theradicals defined above under
 1. to
 7. are unsubstituted or,independently of one another, mono- to trisubstituted by a) —OH, b)halogen such as fluorine, chlorine, bromine or iodine, c) —NO₂, d)—C(O)—O—(C₁-C₂₀)-alkyl in which alkyl is linear or branched andunsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino ornitro, or e) —(C₆-C₁₄)-heterocycle which is unsubstituted or mono- totrisubstituted by halogen, hydroxyl, amino or nitro, said methodcomprising a) a racemic mixture comprising the compound of the formulaII, alcohol dehydrogenase, water, cofactor NADP or NAD and an organicsolvent having a logP of from 0.6 to 1.9, from the series diethyl ether,tert-butyl methyl ether, diisopropyl ether, ethyl acetate, b) isincubated in a two-phase system of water and organic solvent immisciblewith water and C) the enantiomerically pure (S)-hydroxy compound isisolated.
 23. The method as claimed in claim 22, wherein acetone isadded.